In the developing embryo, the primary vascular network is established by in situ differentiation of mesodermal cells in a process called vasculogenesis. It is believed that all subsequent processes involving the generation of new vessels in the embryo and neovascularization in adults, are governed by the sprouting or splitting of new capillaries from the pre-existing vasculature in a process called angiogenesis (Pepper et al., 1996, Enzyme & Protein, 49:38-162; Breier et al., 1995, Dev. Dyn., 204:228-239; Risau, 1997, Nature, 386:671-674). Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Modulation, regulation and/or stabilization of angiogenesis is useful in preventing or alleviating various pathological processes.
On the other hand, promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans.
The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. The molecular mechanisms underlying the complex angiogenic processes are far from being understood.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFα), and hepatocyte growth factor (HGF). See for example Folkman et al., 1992, J. Biol. Chem., 267:10931-10934 for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors are primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs).
The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cysteine knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., 1996, The EMBO Journal, 15:290-298). VEGF-D binds to both VEGFR-2 and VEGFR-3. VEGF-E binds with high affinity to VEGFR-2 and neuropilin-1, but neither to VEGFR-1 nor to VEGFR-3, inducing vascular permeability and potent angiogenic activity both in vitro and in vivo (Ogawa et al., J. Biol. Chem. 1998. 273: 31273-31282; Meyer et al., EMBO J. 1999. 18: 363-374; Wise et al., Proc. Natl. Acad. Sci. USA 1999. 96: 3071-3076.). A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
The isolation and cloning of a novel 130-135 kDa VEGF isoform specific receptor has been reported in Soker et al., 1998, Cell, 92:735-745. This VEGF receptor was found to specifically bind the VEGF165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., 1998, Cell, 92:735-745). Surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., 1998, J. Biol. Chem., 273:22272-22278).
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., 1992, Oncogene, 8:11-18; Kaipainen et al., 1993, J. Exp. Med., 178:2077-2088; Dumont et al., 1995, Dev. Dyn., 203:80-92; Fong et al., 1996, Dev. Dyn., 207:1-10) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., 1995, Proc. Natl. Acad. Sci. USA, 9:3566-3570). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., 1995, Nature, 376:66-70). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., 1998, Proc. Natl. Acad. Sci. USA, 95:9349-9354). The reasons underlying these differences remain to be explained but suggest that receptor signaling via the tyrosine kinase is not required for the proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., 1995, Nature, 376:62-66; Shalaby et al., 1997, Cell, 89:981-990). Inactivation of VEGFR-3 results in cardiovascular failure due to abnormal organization of the large vessels (Dumont et al., 1998, Science, 282:946-949).
Although VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., 1995, Nature, 376:66-70). It is also is expressed by most, if not all, vessels in embryos (Breier et al., 1995, Dev. Dyn., 204:228-239; Fong et al., 1996, Dev. Dyn., 207:1-10). In adults, monocytes and macrophages also express this receptor (Barleon et al., 1996, Blood, 87:3336-3343).
The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development, but as embryogenesis proceeds, it becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., 1994, Cancer Res., 54:6571-6577; Kaipainen et al., 1995, Proc. Natl. Acad. Sci. USA, 92:3566-3570). VEGFR-3 continues to be expressed on lymphatic endothelial cells in adults. This receptor is essential for vascular development during embryogenesis. Targeted inactivation of both copies of the VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5. On the basis of these findings it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., 1996, EMBO J., 15:290-298).
PDGF plays an important role in the growth and/or motility of connective tissue cells, fibroblasts, myofibroblasts and glial cells (Heldin et al., “Structure of platelet-derived growth factor: Implications for functional properties”, 1993, Growth Factor, 8:245-252). In adults, PDGF stimulates wound healing (Robson et al., 1992, Lancet, 339:23-25). Structurally, PDGF isoforms are disulfide-bonded dimers of homologous A- and B-polypeptide chains, arranged as homodimers (PDGF-AA and PDGF-BB) or a heterodimer (PDGF-AB).
PDGF isoforms exert their effects on target cells by binding to two structurally related receptor tyrosine kinases (RTKs). The alpha-receptor binds both the A- and B-chains of PDGF, whereas the beta-receptor binds only the B-chain. These two receptors are expressed by many cell lines grown in vitro, and are mainly expressed by mesenchymal cells in vivo. The PDGFs regulate cell proliferation, cell survival and chemotaxis of many cell types in vitro (reviewed in Heldin et al., 1998, Biochim. Biophys. Acta, 1378:F79-113). In vivo, they exert their effects in a paracrine mode since they often are expressed in epithelial (PDGF-A) or endothelial cells (PDGF-B) in close apposition to the PDGFR expressing mesenchyme. In tumor cells and in cell lines grown in vitro, coexpression of the PDGFs and the receptors generate autocrine loops which are important for cellular transformation (Betsholtz et al., 1984, Cell, 39:447-57; Keating et al., 1990, J. R. Coll Surg Edinb., 35:172-4). Overexpression of the PDGFs have been observed in several pathological conditions, including malignancies, arteriosclerosis, and fibroproliferative diseases (reviewed in Heldin et al., 1996, The Molecular and Cellular Biology of Wound Repair, New York: Plenum Press, 249-273).
The importance of the PDGFs as regulators of cell proliferation and survival are well illustrated by recent gene targeting studies in mice that have shown distinct physiological roles for the PDGFs and their receptors despite the overlapping ligand specificities of the PDGFRs. Homozygous null mutations for either of the two PDGF ligands or the receptors are lethal. Approximately 50% of the homozygous PDGF-A deficient mice have an early lethal phenotype, while the surviving animals have a complex postnatal phenotype with lung emphysema due to improper alveolar septum formation because of a lack of alveolar myofibroblasts (Boström et al., 1996, Cell, 85:863-873). The PDGF-A deficient mice also have a dermal phenotype characterized by thin dermis, misshapen hair follicles and thin hair (Karlsson et al., 1999, Development, 126:2611-2). PDGF-A is also required for normal development of oligodendrocytes and subsequent myelination of the central nervous system (Fruttiger et al., 1999, Development, 126:457-67). The phenotype of PDGFR-alpha deficient mice is more severe with early embryonic death at E10, incomplete cephalic closure, impaired neural crest development, cardiovascular defects, skeletal defects, and edemas (Soriano et al., 1997, Development, 124:2691-70). The PDGF-B and PDGFR-beta deficient mice develop similar phenotypes that are characterized by renal, hematological and cardiovascular abnormalities (Leveen et al., 1994, Genes Dev., 8:1875-1887; Soriano et al., 1994, Genes Dev., 8:1888-96; Lindahl et al., 1997, Science, 277:242-5; Lindahl, 1998, Development, 125:3313-2), where the renal and cardiovascular defects, at least in part, are due to the lack of proper recruitment of mural cells (vascular smooth muscle cells, pericytes or me sangial cells) to blood vessels (Levéen et al., 1994, Genes Dev., 8:1875-1887; Lindahl et al., 1997, Science, 277:242-5; Lindahl et al., 1998, Development, 125:3313-2).
A member of the PDGF family of growth factors is PDGF-D, which is described in International patent application no. WO 00/27879 and published US application no. 2002/0164710 A1, the entire contents of which are incorporated herein by reference. PDGF-D has the ability to stimulate, or enhance, or both, one or more of proliferation, differentiation, growth, and motility of cells expressing a PDGF-D receptor. Cells affected by PDGF-D include, but are not limited to, endothelial cells, connective tissue cells, myofibroblasts and glial cells. PDGF-D and compositions containing it are useful for various therapeutic applications involving the modulation, regulation and/or stabilization of angiogenesis, and particularly for the treatment of edemas which result from leaky vessels.
PDGF-D is structurally homologous to PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C and VEGF-D. The polynucleotide sequence of at least nucleotides 935 to 1285 set out in FIG. 2 (SEQ ID NO:1) encodes a portion of the PDGF/VEGF homology domain, which is the bioactive fragment of PDGF-D. The bioactive fragment is also included in the 200 amino acids set out in FIG. 3 (SEQ ID NO:3) (excluding the first 24 amino acid residues, which are due to a cloning artifact) or the 322 amino acid sequence set out in FIG. 4 (SEQ ID NO:4).
PDGF-D has the ability to stimulate one or more of proliferation, differentiation, motility, survival or vascular permeability of cells expressing a PDGF-D receptor including, but not limited to, vascular endothelial cells, lymphatic endothelial cells, connective tissue cells (such as fibroblasts), myofibroblasts and glial cells. PDGF-D also has the ability to stimulate wound healing. A preferred fragment is a truncated form of PDGF-D comprising a portion of the PDGF/VEGF homology domain (PVHD) of PDGF-D. The portion of the PVHD is from residues 254-370 of FIG. 1 (SEQ ID NO:2) where the putative proteolytic processing site RKSK starts at amino acid residue 254 (SEQ ID NO:2). However, the PVHD extends toward the N terminus up to residue 234 of FIG. 1 (SEQ ID NO:2). Herein the PVHD is defined as truncated PDGF-D. The truncated PDGF-D is the putative activated form of PDGF-D.
As indicated above, there are numerous clinical situations where angiogenesis is desired to be promoted, and methods have been proposed using one of the numerous members of the VEGF/PDGF family of growth factors known to have angiogenesis stimulation effects. For example, VEGF has been shown to be intimately involved in the entire sequence of events leading to growth of new blood vessels. Gross et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Folkman et al., Proc. Nat'l. Acad. Sci., 76(10): 5217-21 (1979). Five human VEGF isoforms of 121, 145, 165, 189 and 206 amino acids have been isolated. Gross, et al., Proc. Nat'l. Acad. Sci., 80(9): 2623-27 (1983), Leung, et al., Science, 246: 1306-09 (1989), Poltorak et al., J. Biol. Chem., 272(11): 7151-78 (1997). Among the isoforms, VEGF 165 seems to be the most effective and most commonly used. The effect of VEGF 165 in augmenting perfusion and in stimulating formation of collateral vessels has been shown in animal models Hopkins et al., J. Vascular Surgery, 27(5): 886-94 (1998), Asahara et al., Circulation, 91(11): 2793-801 (1995), Hariawala et al., J. Surg. Res., 63(1): 77-82 (1996), Bauters et al., Circulation, 91(11): 2802-9 (1995), Bauters et al., Am. J. Physiol., 267(4 Pt 2): H1263-71 (1994), Takeshita et al.,. J. Clin. Invest., 93(2): 662-70 (1994), Takeshita, et al., Circulation, 90(5 Pt 2): II228-34 (1994), Takeshita, et al., Am. J. Path., 147(6): 1649-60 (1995), Banai, et al., Circulation, 89(5): 2183-9 (1994). In clinical trials, successful induction of collateral blood vessels in ischemic heart disease and critical limb ischemia by VEGF have also been reported. Baumgartner et al., Circulation, 97(12): 1114-23 (1998), Losordo, et al., Am. Heart J., 138(2 Pt 2): 132-41 (1999).
Angiogenesis, however, is a complex process that includes activation, migration and proliferation of endothelial cells and formation of new blood vessels. D'Amore, et al., Ann. Rev. Physiol., 49(9-10): 453-64 (1987). The process likely requires a network of members of the VEGF/PDGF family of growth factors, and use of a single factor alone in promoting angiogenesis may have undesired or unsatisfactory results. For example, it is known that the vascular endothelial growth factor-A (“VEGF”) causes both abnormal blood vessel growth (angiogenesis) and blood vessel leakage in the eye. Specifically, preclinical studies have shown that a) in multiple animal species, including humans, VEGF levels are elevated around growing and leaky blood vessels, b) blocking VEGF results in the prevention and regression of these abnormal vessels in primates and other species and c) VEGF alone is sufficient to trigger the abnormal blood vessel growth and blood vessel leakage that characterizes wet age-related macular degeneration (AMD). See A. P. Adamis et al., Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate, 114(1) Arch. Ophthalmol. 66-71 (1996); A. Kvanta et al., Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor, 37 Invest. Ophthalmol. Vis. Sci. 1929-34 (1996); G. Lutty et al., Localization of VEGF in human retina and choroids, 114 Arch. Ophthalmol. 971-77 (1996); M. J. Tolentino et al., Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate, 103(11) Ophthalmology 1820-28 (1996); M. J. Tolentino, Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate, 114(8) Arch. Ophthalmol. 964-70 (1996). To minimize the undesired effect of VEGF, VEGF antagonists have been proposed, but these antagonists are obviously not suitable for situations where stimulation of angiogenesis is desired.
Thus, there is a significant need to be able to modulate the angiogenesis promoting activities of one member of the PDGF/VEGF family of growth factors, preferably with another member of the family.